Chemotaxonomic Significance of Xanthomonadins

Xanthomonadins are a unique class of carotenoid-like pigments produced by members of the phytopathogenic genus Xanthomonas. These pigments are brominated, aryl-polyene, yellow, water-insoluble pigments that are associated exclusively with the outer membrane of the bacterial cell wall (17). Differentiated into 15 groups according to the number of bromine atoms, the absorption maxima and mass spectrometric M + value (6), and methylation (15), these pigments are useful chemotaxonomic markers for Xanthomonas (16). Other yellow pigmented bacteria that are either taxonomically similar or distant do not produce xanthomonadins. All yellow Xanthomonas spp. produce xanthomonadins (16).

The yellow color of xanthomonadins was recognized early as a significant characteristic of the genus (5). Mortimer Starr first demonstrated the carotenoid nature of this yellow coloring (14). Subsequent comparative studies (16; see comparative list) showed that all yellow pigmented strains of Xanthomonas produced pigments with similar chromatographic and spectral absorption properties. In 1973, Andrewes and Andrewes et al. (1, 2) determined that these pigments consisted of mixtures of brominated, aryl-polyene esters. Thus, xanthomonadins and carotenoids, while chemically different, do share the trait of having polyene chains (8). In 1976, Andrewes et al. (3) published the exact structure of xanthomonadin I, a dibrominated xanthomonadin from Xanthomonas juglandis (see Figure 1). It became clear by 1977 that xanthomonadins from different xanthomonads differed in bromination and methylation (15). Studies with X. albilineans, X. arboricola pv. pruni, X. axonopodis, X. arboricola pv. juglandis,X. campestris, X. fragariae, X. hyacinthi, X. axonopodis pv. phaseoli, X. translucens, X. vesicatoria and X. ampelina (currently Xylophilus ampelinus, see 18) indicated that the bromination and methylation patterns of xanthomonadins were useful for identification of members within the genus. In addition, Starr et al. (15) showed distinctive electronic absorption and chemical properties of the pigments from Xylophilus ampelinus, suggesting that this species does not belong in the genus Xanthomonas.

Purification of Xanthomonadins

Isolations from plant tissues and soil debris often yield many yellow-pigmented bacteria. It may be difficult to distinguish colonies of Xanthomonas spp. visually from colonies of saprophytic bacteria by color, especially with Xanthomonas species that do not produce copious amounts of extracellular polysaccharide (EPS). Because the yellow xanthomonadin pigments of Xanthomonas are unique to the genus, pigment extraction and chromatographic analysis can be an important tool for bacterial genus identification (7). Additional chromatographic, adsorption, and analytical spectroscopy data for species characterization are available (see 15, 16).

The following procedure for identification of xanthomonadins (7) is from the Laboratory Guide for the Identification of Phytopathogenic Bacteria, 3rd edition.

Streak a colony onto nutrient agar (nutrient agar should not contain an additional carbohydrate because the resulting copious slime will interfere with chromatography of the pigments). (A known xanthomonad should be included as a control.)

After 48 h growth, scrape the bacteria from the surface and add to 3 ml of spectrophotometry grade methanol in a test tube with a screw cap. Enough bacteria should be added to the methanol to give a turbidity equivalent to near 1010 CFU/ml, (approximately 0.5 OD at 600 nm).

Place the capped tube in a boiling water bath until the pigment has been removed from the bacteria (solution becomes yellow).

Centrifuge at 1,000 g for 15 min to remove cell debris.

Decant supernatant and evaporate the methanol extract in a water bath at 50-60o C until the optical density of the pigment extract reaches 0.4 at 443 nm.

Spot five 5 nl aliquots on a precoated, thin-layer chromatography sheet of silica gel 60 of 0.2 mm thickness (E. Merck, Darmstadt, W. Germany). Apply a total of 25 nl per spot, allowing each 5 nl amount to dry before applying the next.

Place plate in developing apparatus with anhydrous spectrophotometry grade methanol as the solvent. Allow the solvent front to move approximately 10 cm.

Outline the yellow spots with a pencil when the silica gel is still wet. A yellow spot with an average Rf value of 0.45 (range of 0.42 to 0.49) is positive for xanthomonadins.

Genetics of Xanthomonadin Production

Xanthomonadin production is controlled by a gene cluster consisting of seven transcriptional units (12, Figure 2) and is regulated by an extracellular bacterial pheromone (4, 11). The gene cluster appears to be present in all xanthomonads (including the white variant X. manihotis) (10) and in the recently reclassified Stenotrophomonas (Xanthomonas) maltophilia (Chun, personal communication). Within the gene cluster is pigB, which is required for the production of a diffusible factor (DF). The diffusible factor was shown to be a butyrolactone (4, Figure 3) by mass spectroscopic analysis and functions similarly to autoregulatory signals found in other bacteria. DF production is essential for both xanthomonadin and EPS production by the bacterium (11). Two transcriptional units: xanB1 which encodes a putative reductase/halogenase; and xanB2 which encodes a putative pteridine-dependent dioxygenase-like protein (13) reside in pigB. Of these, only xanB2 is required for DF production. Hence, single site pigB (xanB2) mutants are typically white and produce less EPS (Figure 4). The remaining genes within the cluster appear to be related to the production and modification of the xanthomonadin.

Biological Role of Xanthomonadins

Xanthomonadins were shown to play a role in protection against damage by visible light in the presence of oxygen (photodynamic damage) in X. juglandis (8) and X. campestris pv. campestris (Poplawsky and Chun, personal comm.). Pheromone, xanthomonadin and extracellular polysaccharide production may be required for epiphytic survival by X. c. pv. campestris on crucifer leaves (4, 9). It is well known that epiphytic survival is essential for Xanthomonas bacteria to establish a successful infection. When both xanthomonadin and EPS production are deficient, populations of X. c. pv. campestris are as much as 1000-fold lower in planta (4). This resulted in significantly fewer lesions (from 8.7 to 1.7 lesions per leaf) on spray-inoculated crucifer leaves (Figure 5).

Summary

Xanthomonadins play a major role in defining the genus Xanthomonas and can be used as supportive data for speciation within the genus. Aside from their bromine scavenging ability (15), these pigments are essential for the pathogenic success of the bacterium (see related images below). Without the protective action of the polyene chain, the bacteria would be limited in their ability to survive epiphytically. Thus, successful entry and establishment of an infection in plant tissues would be drastically diminished.

2 Color refers to Starr and Stephens determination of "zones" in order of appearance from top to bottom on magnesia-Celite columns (see Starr, M. P. and Stephens, W. L. 1964. Pigmentation and taxonomy of the genus Xanthomonas. J. Bacteriol. 87:293-302).

3 Parentheses around an absorption maximum indicate a shoulder or broad central peak centered around that approximate wavelength